Genome-wide analysis and functional characterization of CHYR gene family associated with abiotic stress tolerance in bread wheat (Triticum aestivum L.)

Background CHY zinc-finger and RING finger (CHYR) proteins have been functionally characterized in plant growth, development and various stress responses. However, the genome-wide analysis was not performed in wheat. Results In this study, a total of 18 TaCHYR genes were identified in wheat and classified into three groups. All TaCHYR genes contained CHY-zinc finger, C3H2C3-type RING finger and zinc ribbon domains, and group III members included 1–3 hemerythrin domains in the N-terminus regions. TaCHYR genes in each group shared similar conserved domains distribution. Chromosomal location, synteny and cis-elements analysis of TaCHYRs were also analyzed. Real-time PCR results indicated that most of selected 9 TaCHYR genes exhibited higher expression levels in leaves during wheat seedling stage. All these TaCHYR genes were up-regulated after PEG treatment, and these TaCHYRs exhibited differential expression patterns in response to salt, cold and heat stress in seedling leaves. The growth of yeast cells expressing TaCHYR2.1, TaCHYR9.2 and TaCHYR11.1 were inhibited under salt and dehydration stress. Moreover, gene ontology (GO) annotation, protein interaction and miRNA regulatory network of TaCHYR genes were analyzed. Conclusions These results increase our understanding of CHYR genes and provide robust candidate genes for further functional investigations aimed at crop improvement. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-022-03589-7.

. C3H2C3-type RING finger domain, also referred to as RING-H2 finger, exists in the C-terminal region of CHYR protein, that can bind two atoms of zinc and may be involved in mediating protein-protein interactions [4,7]. Besides, group III members, also named as BTS/BTSL (BRUTUS/BRUTUS-like) or HRZ (hemerythrin RING zinc-finger) proteins, which contain N-terminal hemerythrin (HHE) domains and play an essential role in regulating iron homeostasis [8][9][10][11].
CHYR genes have been identified in diverse plants, such as maize (Zea mays), Arabidopsis, rice, and soybean (Glycine max) [2,8,14,18], however, a genome-wide identification of CHYR genes in wheat was not preformed. In this study, a genome-wide analysis of CHYR genes was performed in wheat to characterize their sequences, evolutionary relationships, expression patterns and stress tolerance in yeast cells under various abiotic stress treatments. These results will provide a valuable foundation for further functional studies of TaCHYR genes under abiotic stress.

Characteristics and phylogenetic analysis of TaCHYRs in T. aestivum
A total of 18 CHYR genes, encoding 50 transcripts, were identified from a wheat genome based on a Hidden Markov Model (HMM) search against of a CHY zinc-finger domain (PF05495) ( Table S1). According to the chromosome position and phylogenetic relationship of TaCHYR genes, we named them from TaCHYR1 to TaCHYR18. These 18 predicted TaCHYR genes encoded polypeptides of 245 (TaCHYR7.2) to 1245 (TaCHYR17.8) amino acid residues with the putative molecular weights ranging from 28.03 to 140.21 kDa. The isoelectric point (pI) value varied from 5.59 (TaCHYR16.5) to 7.55 (TaCHYR9.2), and the calculated grand average of hydrophilic index (GRAVY) was from -0.166 (TaCHYR6.2 and TaCHYR6.3) to -0.484 (TaCHYR11.2), suggesting that these 18 TaCHYR genes were hydrophilic proteins. In addition, the subcellular localization prediction indicated that TaCHYR proteins were all located in the nucleus.
To further investigate the phylogenetic relationship of TaCHYRs, AtCHYRs and GmCHYRs were also used to construct a phylogenetic tree ( Fig. 1 and Table S2), the results indicated that TaCHYR genes were divided into 3 subfamilies: group I, II, and III ( Fig. 1 and Fig. 2A), which was consistent with previous phylogenetic analyses of plant CHYR genes [2]. Group I (TaCHYR1-6), group II (TaCHYR7-12) and group III (TaCHYR13-18) subfamilies included 6 members in wheat, respectively ( Fig. 1 and Table S1).

Gene structure and conserved motifs analysis of TaCHYR genes
To investigate the structural characteristics of TaCHYR genes, the exon-intron structures and conserved motifs of 18 TaCHYR genes were analyzed (Fig. 2). The TaCHYR genes contained 10-15 exons, the coding sequence (CDS) of group III members were longer than that of other two groups, and most genes with closer evolutionary relationships had similar exon-intron structures (Fig. 2B). Furthermore, the conserved domains of TaCHYRs were analyzed, the results showed that all TaCHYRs contained CHY-zinc finger, C3H2C3-type RING finger and zinc ribbon domains ( Fig. 2C and Fig. 3). These results further verified the reliability of the identified TaCHYR gene family members. Besides, group III members contained 1-3 hemerythrin domains in the N-terminus. Meanwhile, we predicted the conserved motifs of TaCHYR gene family using MEME tools ( Fig. 2C and Fig. S1). Among the detected 10 motifs, motif 2, 4 and 6 formed CHY-zinc finger domain, motif 3 and 9 made up RING finger domain. Zinc ribbon domain consisted of motif 7 and 9. Hmerythrin domain included motif 8 and 10. Additionally, motif 1, 5 and 10 were unique to group III members. These results indicated that the composition of conserved motifs were varied among different CHYR subfamilies, but TaCHYRs with closer evolutionary relationships had more similar conserved domains.

Chromosomal location, synteny and Ka/Ks analysis of TaCHYRs
The chromosomal location and synteny of TaCHYR gene family were analyzed according to their genomic   Fig. S2). TaCHYR genes were distributed on chromosome 1, 3, 4, and 5. Eighteen TaCHYR genes were evenly distributed among A, B, and D subgenomes: TaCHYR1, 4, 7, 10, 13 and 16 were located on A subgenome; TaCHYR2, 5,8,11,14 and 17 on B subgenome; TaCHYR3, 6,9,12,15 and 18 on D subgenome. Chromosome 1A, 1B, 1D, 3A, 3B and 3D contained two TaCHYR genes, respectively. Chromosome 4A, 4B, 4D, 5A, 5B and 5D contained only one TaCHYR genes, respectively (Fig. S2). A total of 22 paralogous TaCHYR gene pairs were determined in wheat genome, which all undergone WGD (whole genome duplications) or segmental duplication events ( Fig. 4 and Table S3). Eighteen genes were all involved in gene duplication events, and these results indicated that TaCHYR genes might be produced by segmental duplication events and these duplication events might contribute to the evolution of TaCHYR genes. Moreover, the non-synonymous (Ka) and synonymous substitution (Ks) ratios were calculated to investigate selective pressure of these 22 paralogous gene pairs (Table S3). The Ka/Ks ratios of paralogous gene pairs were all less than 1, indicating these TaCHYR genes were under strong purifying selection to maintain the function of TaCHYR gene family.

Cis-elements analysis of TaCHYR genes
The variable cis-elements in the gene promoters might indicate that these genes perform different functions in plant growth, development and various stress responses. To further investigate the functions of TaCHYR genes, 1.5 kb of the upstream region of each TaCHYR gene transcription start site (TSS) was extracted and then analyzed by using the PlantCARE database to identify and count the cis-elements (Fig. 6). These cis-elements were divided into four categories, i.e., stress responsive-, light responsive-, hormone responsive-, and growth and development related-elements. The promoter of TaCHYR genes included masses of stress and hormone responsive-elements, especially DRE (drought-responsive element), MBS (MYB binding site), MYC and STRE (stress-responsive element) among stress responsive-elements, and ABRE (ABA-responsive element) and CGTCA-motif (MeJA-responsive element) among hormone responsiveelements. Additionally, group I members had abundant ARE (anaerobic induction-responsive element). G-box abounded in TaCHYR promoters, which could interact with bZIP or bHLH transcription factors to participate in biological processes [19]. These results indicated that the TaCHYR genes might play significant roles in plant growth, development and respond to various stresses in wheat.

GO annotation and network construction of TaCHYRs
To further understand the function of TaCHYR from molecular levels, all TaCHYR proteins were annotated by gene ontology (GO) (Fig. 7A and Table S6). These TaCHYR proteins were assigned with 22 GO terms belonging to the cellular component, molecular function and biological process (Fig. 7A). Under the cellular component category, most TaCHYR proteins (74%) were located in the nucleus (GO:0005634). Under the molecular function category, all TaCHYRs were involved in . Under the biological process category, most TaCHYRs were involved in protein metabolic process (GO:0019538), protein modification process (GO:0036211), protein catabolic process (GO:0030163), and homeostatic process (GO:0042592). According to protein-protein interactions (PPIs) analysis, TaCHYR proteins could bind other TaCHYR family members or other E3 ubiquitin-protein ligases to form heterodimers ( Fig. 7B and Table S7).

Expression patterns of TaCHYR Genes in different tissues
To investigate the tissue-specific expression patterns of the TaCHYR genes, the gene expression levels of 9 selected TaCHYR genes belonging to group I (TaCHYR1, 2 and 4), group II (TaCHYR8, 9, 11 and 12) and group III (TaCHYR13 and 17) members were determined by using real-time PCR in roots, stems, and leaves tissues during wheat seedling stage (Fig. 8). Most of selected 9 TaCHYR genes exhibited higher expression levels in leaves, such as TaCHYR1, 4,8,13, and 17 were highly expressed in leaves. Group I (TaCHYR1, 2 and 4) and group III (TaCHYR13 and 17) members were predominantly expressed in leaves and stems, followed by roots. Group II (TaCHYR8, 9, 11 and 12) members showed differential expression profiles in roots, stems, and leaves tissues. TaCHYR8 and TaCHYR9 showed highest expression levels in leaves and roots, respectively. TaCHYR11 and TaCHYR12 exhibited the highest expression levels in stems, followed by leaves, and finally in roots. In particular, most paralogous genes showed similar expression patterns, e.g., TaCHYR1/2, TaCHYR11/12, and TaCHYR13/17. However, the paralogous genes TaCHYR8/9 had different expression profiles in various tissues, indicating TaCHYR8 and 9 might have diverse function due to ecological or environmental adaptation. These might be caused by modification in their promoters, some special regulatory elements, or functional segregation with a long-term evolutionary process.

Expression patterns of TaCHYR genes under abiotic stress
To insight into the function of TaCHYR genes under abiotic stress, real-time PCR was used to determine the expression patterns of these 9 TaCHYR genes under PEG, salt and heat and cold stress in leaves during the wheat seedling stage (Fig. 9). Under PEG stress, all these TaCHYR genes were up-regulated and reached the highest expression level at 36 h. TaCHYR11 and TaCHYR12 were significantly up-regulated more than 65-fold and 21-fold under PEG stress compared with the control, respectively (Fig. 9A). These TaCHYRs exhibited differential expression patterns in seedling leaves in response to salt, cold and heat stress. In salt stress condition, TaCHYR2 and 4 were obviously up-regulated and reached the highest expression level at 36 h, and TaCHYR8 and 9 were significantly down-regulated (Fig. 9B). After heat stress treatment, most these TaCHYR genes were downregulated compared with control, except TaCHYR4 was up-regulated (Fig. 9C). Under cold stress, TaCHYR2, 4, 11, 12, 13 and 17 were obviously up-regulated, and the expression of TaCHYR1, 8 and 9 were significantly suppressed compared with control (Fig. 9D). Moreover, the paralogous genes had similar expression patterns, e.g., TaCHYR11/12 had almost consistent expression patterns under PEG, salt, heat or cold stress. These results suggested that TaCHYR genes had different response to different stress and play various regulatory roles in abiotic stress resistance.

Negative regulation of dehydration and salt stress tolerance in recombinant yeast cells
According to the expression profiles, three TaCHYR genes (TaCHYR2.1, TaCHYR9.2, and TaCHYR11.1) were selected to clone into pGADT7 vector, and then transformed into the osmotic-sensitive yeast mutant BY4741 (Δhog1) to verify the ability in response to stress tolerance in yeast mutant cells (Fig. 10). The results suggested that the growth of the Δhog1 yeast cells containing these recombinant vectors (pGADT7-TaCHYR2.1, pGADT7-TaCHYR9.2 or pGADT7-TaCHYR11.1) were inhibited Fig. 7 Gene ontology (GO) annotation A and protein-protein interactions B and miRNA targeting C analysis of TaCHYR genes. A The GO terms were showed on the X axis, and the number of genes were showed on the Y axis. C Pink and yellow boxes indicated TaCHYR genes and miRNAs in wheat, respectively in YPD medium containing NaCl (0.4 and 0.6 M) and D-Sorbitol (1.0 and 1.2 M) compared with the control (pGADT7 empty vector), and the growth of yeast cells were inhibited more obviously with the increase of NaCl and D-Sorbitol concentration ( Fig. 10A and 10B). Moreover, the growth of yeast expressing TaCHYR2.1 was severely inhibited in salt and dehydration treatment conditions. The growth of yeast expressing TaCHYR9.2 was similar to TaCHYR11.1, which was slightly inhibited under salt and dehydration stress. These results revealed that TaCHYR2.1, TaCHYR9.2 and TaCHYR11.1 might participate in protein catabolic and homeostatic process under salt and dehydration stress in wheat.

Discussion
Previously, CHYR genes have been identified in diverse plants, such as maize, Arabidopsis, rice and soybean [2,8,14,18]. However, CHYR genes have not been genomewide identified in wheat. In this study, 18 CHYR genes were identified in wheat and classified into 3 groups (Fig. 1). All TaCHYR proteins contained CHY-zinc finger, C3H2C3-type RING finger and zinc ribbon domains (Fig. 2C). Besides, there were 1-3 hemerythrin domains in the N-terminus of group III members, which may play vital roles in regulating iron homeostasis [8]. Previous studies showed that CHYR genes occurred during evolution of green plants, group I members were present in Bryophyta, Pteridophyta and Gymnosperms, while group II members were absent, suggesting the differentiation of CHYRs between group I and II members might occur after angiosperm differentiation [2]. Therefore, group I and II members could be found to coexist in monocotyledonous (wheat and rice) and dicotyledonous (Arabidopsis and soybean) plants ( Fig. 1  and Fig. 5). Group III members were far away from the other two groups in plants evolution process, indicating that they might originated from different ancestors [2]. In this study, we also analyzed the synteny relationships of CHYR genes between the wheat and other plant species, three orthologous gene pairs that located on the same chromosomes between wheat A subgenome and T. urartu were identified. Similarly, six orthologous gene pairs which located on the same chromosomes between wheat D subgenome and Ae. tauschii were identified ( Fig. 5 and Table S4). These TaCHYR genes might be originated from orthologous genes in T. urartu and Ae. tauschii with the occurrence of natural hybridization events. Moreover, 22 paralogous gene pairs were identified among 18 TaCHYR genes in wheat genome, which all undergone WGD or segmental duplication events and a strong purifying selection pressure ( Fig. 4 and Table S3). These results indicated that WGD or segmental duplications played crucial roles in the expansion of the TaCHYR genes.
CHYR genes participated in various biological processes to play vital roles in plant growth, development and various stress responses [3]. Real-time PCR results indicated that most of selected 9 TaCHYR genes exhibited higher expression levels in leaves during wheat seedling stage (Fig. 8), suggesting these TaCHYRs might play an important function in leaves. The tissuespecific expression of TaCHYR genes mainly related with the variable cis-elements present in its promoters (Fig. 6). Under PEG stress, all TaCHYR genes were upregulated, which might be regulated by masses of stress and hormone responsive-elements in the TaCHYR promoter regions (Fig. 6 and Fig. 9A). Moreover, the similar expression patterns were detected in the paralogous genes, e.g., TaCHYR11/12 were significantly induced more than 65-fold and 21-fold compared with the control after PEG treatment, respectively. However, less stress and hormone responsive-elements were identified in the promoter of TaCHYR11, probably due to the lack of the 425 bp genomic sequences in TaCHYR11 promoter region (Fig. 6 and Fig. S3). Therefore, we speculated that TaCHYR genes played vital roles when plants suffered dehydration stress. MiRNAs also played an important role in regulating the expression of downstream genes [21]. Here, 21 miRNAs were identified, and tae-miR9657a-3p targeted 6 TaCHYR genes, implying tae-miR9657a-3p played a key role in regulating the expression of TaCHYR genes (Fig. 7C and Table S8).
GO annotation analysis indicated TaCHYRs were E3 ubiquitin-protein ligases (Fig. 7A and Table S6), and regulated the plant adaptive response to abiotic stress via ABA-mediated signaling, and especially through modulating the stability of bZIP and bHLH transcription factor [19,22]. Previous studies have showed that CHYR1 promoted ABA-induced stomatal closure, reactive oxygen species production, and plant drought tolerance in Arabidopsis, the similar results were also found in poplar [3,13]. CHYR gene CaASRF1 positively modulates ABA signaling and the drought stress response via modulation of bZIP transcription factor CaAIBZ1 stability [22]. In contrary, the rice E3 ubiquitin-protein ligase droughtinduced SINA protein 1 (OsDIS1) negatively regulated drought stress response through transcriptional or post-translational regulation of various stress-related genes [23]. Rice OsRZFP34 gene enhances stomatal opening, leaf cooling and ABA insensitivity [14]. Arabidopsis E3 ubiquitin ligase PUB11 negatively regulated drought tolerance by degrading the receptor-like protein kinases LRR1 and KIN7 [24]. The C3HC4-type RING finger E3 ubiquitin ligase TaSADR1 negatively regulates drought resistance in transgenic Arabidopsis [25]. In our study, the growth of yeast cells expressing TaCHYR2.1, TaCHYR9.2 and TaCHYR11.1 were inhibited in salt and dehydration treatment conditions, probably caused by the ubiquitination of interacting proteins resulting in degradation of stress-related proteins. In the future, these genes would be knocked out through CRISPR technology to obtain drought-resistant wheat. These results still require to be confirmed in wheat, and E3 ubiquitinprotein ligase activity of TaCHYRs also require further experimental verification.

Conclusions
In this study, 18 TaCHYR genes were identified, which can be divided into three groups. All TaCHYR genes contained CHY-zinc finger, C3H2C3-type RING finger and zinc ribbon domains, and group III members included 1-3 hemerythrin domains. TaCHYR genes were distributed on chromosome 1, 3, 4, and 5, and evenly distributed among A, B, and D subgenomes. Ka/Ks analysis showed that the TaCHYRs undergone a strong purifying selection pressure during the evolution process. Twentytwo paralogous gene pairs were identified in wheat, and 15, 22, 22 and 20 orthologous gene pairs were identified between wheat with T. urartu, Ae. tauschii, B. distachyon and O. sativa, respectively. The promoters of TaCHYR genes contained masses of stress and hormone responsive-elements. Real-time PCR results suggested that most of selected 9 TaCHYR genes exhibited higher expression levels in leaves during wheat seedling stage. All these TaCHYR genes were up-regulated after PEG treatment, and these TaCHYRs exhibited differential expression profiles in response to salt, cold and heat stress. The growth of yeast cells overexpressing TaCHYR2.1, TaCHYR9.2 and TaCHYR11.1 was suppressed under salt and dehydration stress. Moreover, gene ontology (GO) annotation, protein interaction and miRNA regulatory network of TaCHYR genes were analyzed. These results provide useful information for further functional studies of TaCHYR genes, and lay a foundation to improve wheat quality traits in molecular breeding under abiotic stress.

Phylogenetic relationships, gene structures and domains analysis
The phylogenetic tree was constructed by the neighborjoining (NJ) method with 1000 bootstrap replicates using MEGA11 software and Evolview online service [28,29]. The amino acid sequences of AtCHYRs and GmCHYRs were obtained from a previous report [28,29]. The exonintron structures were identified by comparing CDS and genomic DNA sequences using TBtools [26]. The conserved domains and motifs were annotated using the SMART database (http:// smart. embl. de/) and MEME online server (http:// meme-suite. org/ index. html).

Chromosomal location, synteny and Ka/Ks analysis
The position of TaCHYR genes on chromosomal were obtained according to wheat genome annotation data and then marked on the chromosomes by using the TBtools and circos [26,30]. Multiple collinear scanning toolkits (MCScanX) were used to detect the gene replication events [31]. TBtools was used to determine the Ka (non-synonymous rate), Ks (synonymous rate), and Ka/Ks ratios of the syntenic gene pair with the Nei-Gojobori (NG) method [26].

Cis-element analysis in the promoter
The promoter sequences, 1.5 kb upstream sequences of the transcription start site (TSS) of the TaCHYR genes, were acquired from the wheat database, and the cis-elements in the promoters were analyzed using PlantCARE database (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/) [33].

Real-time PCR analysis
Wheat seeds of "Chinese Spring", which were obtained from the Northwest A&F University, were germinated on moist filter paper at 25/18 ℃ (day/night) with a photoperiod of 16 h light/8 h dark at Henan University of Science and Technology on 20 December 2021. For abiotic stress treatment, seedlings grown in hydroponic culture for two weeks were exposed to 20% PEG6000 (w/v), high salinity (300 mM NaCl), high temperature (42 ℃), and cold (4 ℃).
In each treatment, the leaf tissues were collected every 12 h for 36 h, frozen in liquid nitrogen, and stored at − 80 ℃ [34].
RNAiso Plus (Takara) was used to isolate total RNA from each frozen sample, and the first-strand cDNA was synthesized from total RNA (1 μg) by using Prescript III RT ProMix (CISTRO) according to the manufacturer's instructions. The sequence was amplified using gene-specific primers (Table S9) with 2 × Ultra SYBR Green qPCR Mix (CISTRO), and the actin gene was used as an internal control. The real-time PCR cycling parameters were 95 °C for 30 s, followed by 45 cycles at 95 °C for 5 s and 60 °C for 30 s, with a melting curve analysis. All reactions were performed in triplicate to ensure the reproducibility of the results.